Elevated Serum Interleukin-8 Level as a Preferable Biomarker for Identifying Uncontrolled Asthma and Glucocorticosteroid Responsiveness.

Background
To explore the clinical significance of serum interleukin-8 (IL-8) level as a biomarker for uncontrolled asthma in order to improve our understanding of asthma phenotypes and facilitate the development of new therapeutic agents in the future.


Materials and Methods
A total of 246 uncontrolled asthma patients and 50 healthy controls were selected from an outpatient clinic during October 2015 and April 2016. The clinical data were collected, and the levels of IL-8, IL-6, tumor necrosis factor-α (TNF-α), and immunoglobulin (IgE) were measured in peripheral blood via ELISA assay. The level of serum IL-8 was compared between the glucocorticosteroid groups, receiving inhaled corticosteroids (ICs), oral corticosteroids (OCs), and intravenous corticosteroids (GCs), respectively. Changes in the serum IL-8 level were compared between asthmatics with good and poor glucocorticosteroid responsiveness.


Results
The serum IL-8 level in uncontrolled asthmatics (87.45 pg/mL; 5-7500) was significantly higher than that of the healthy controls (10.9 pg/mL; 6.8-39.65; P< 0.001). The increase in the serum IL-8 level above the normal range occurred in 58.13% of uncontrolled asthmatics. The area under curve (AUC) for serum IL-8 level, indicative of uncontrolled asthma, was 0.816 (95% CI, 0.7605 to 0.8721; P< 0.0001), which was greater than the AUC of fractional exhaled nitric oxide (AUC, 0.711; 95% CI, 0.6057 to 0.8153; P= .0188). The serum IL-8 level showed a significant positive relationship with blood neutrophil count (P= 0.0004), neutrophil percentage (P= 0.027), serum TNF-α protein (P< 0.0001), forced expiratory volume/forced vital capacity (FEV1/FVC) ratio (P< 0.05), and rate of FEV1 change after bronchodilation. The level of IL-8 in patients requiring OCs or GCs treatment was significantly higher than that of ICs patients (186 and 235 pg/mL vs. 61 pg/mL; P< 0.0001). The reduction in the serum IL-8 level was more significant in asthmatic patients with good responsiveness (277 pg/mL (65.3-3124) to 67.8 pg/mL (5-1408); P< 0.0001), compared to those with poor responsiveness (218 pg/mL (64.8-7500) vs. 197 pg/mL (56.9-5238); P= 0.49).


Conclusion
The increase in serum IL-8 level can be used as a preferable biomarker to identify asthma status and initial treatment in asthmatics. The change in IL-8 level also reflects the response to glucocorticosteroids in uncontrolled asthma. These exploratory results suggest an association between the pathophysiology, inflammation, and clinical outcomes of asthma. This raises the possibility of developing new agents for IL-8 inhibition and helps provide more precise and personalized asthma care.


INTRODUCTION
Asthma is a common respiratory disease, characterized by chronic airway inflammation dominated by T helper 2 (Th2) cells. Millions of patients still suffer from this incurable disease, and the control rate of asthma is unsatisfactory worldwide according to epidemiological investigations (1,2). Improvement of the overall asthma control and timely and effective alleviation of symptoms are becoming major clinical problems, which need to be addressed urgently. Proper control of the disease depends on both detection of uncontrolled cases and acceptable response to available asthma medicines. Therefore, search for rapid and convenient biomarkers for evaluation of control conditions and prediction of treatment responsiveness should be resolved in clinics abruptly.
Over the past decades, there have been great interests in the role of inflammation in the pathogenesis and pathophysiology of asthma. Accumulating evidence suggests that cytokine alterations and proinflammatory state are associated with asthma (3)(4)(5). However, research on the role of systematic proinflammatory mediators is more limited than studies on airway local inflammation, and the available data originate from case studies with a small number of samples or controversial results.
Interleukin-8 (IL-8) is considered an important chemotactic factor, involving neutrophil recruitment and activation; it is also active on primed eosinophils. It can be secreted by many structural and immune cells, including bronchial epithelial cells, smooth muscle cells, and macrophages. Some studies have reported increased serum levels of IL-8 during asthma attacks and allergic dermatitis (6). IL-8 level also increases in the induced sputum or bronchoalveolar lavage fluid of asthmatics, suggesting the involvement of IL-8 in the pathogenesis of inflammatory and allergic diseases (7,8).
Brown et al. (9) recently reported that serum Th17associated cytokines, including IL-8, were positively associated with difficult-to-control asthma in inner-city African American children. The association of severe asthma with increased IL-8 level in the sputum or bronchoalveolar lavage fluid has been previously reported in adults and children (10,11). The literature suggests that IL-8 plays an important role in refractory neutrophilic asthma and is an indicator of neutrophilic phenotypes in asthma patients.
As a potent proinflammatory cytokine, the function and role of serum IL-8 in adult asthma have not been yet fully examined. The present study was designed to explore the potential of serum IL-8 in determining the disease status and to describe its significance as an indicator of treatment responsiveness in a series of uncontrolled adult asthma patients. This study was conducted in order to deepen our understanding of asthma phenotypes and to provide a framework for the development of new target therapeutic agents in the future.

Subjects
This observational, case-control study was conducted between October, 2015 and April, 2016 in the outpatient clinic of the Department of Respiratory and Critical Care Medicine, Changhai Hospital. A total of 246 uncontrolled asthma patients above 18 years were diagnosed by a physician according to the Global Initiative for Asthma (GINA) criteria. The uncontrolled asthma condition was evaluated according to the GINA standards (12). Patients with an acute attack or at least 3 of the following manifestations in the past 4 weeks were defined as uncontrolled asthma: 1) daytime symptoms more than twice a week; 2) nocturnal awakening due to asthma; 3) relievers needed more than twice a week; and 4) any activity limitations due to asthma (12).

Pulmonary function test (PFT) and fractional exhaled nitric oxide (FeNO) measurements
The bronchial challenge test (BCT) or bronchodilator test is used to diagnose asthma. PFT was performed using a pneumotachograph-based system in accordance with the recommendations of the European Respiratory Society (13). The FeNO level was determined, using the NIOX MINO chemiluminescence analyzer (Aerocrine AB, Solna, Sweden), according to the guidelines established by the American Thoracic Society (ATS) (13). The patients were asked to inhale the maximum amount of air and were then instructed to exhale air into the valve connected to the analyzer. The flow rate (50 mL/s) was kept constant, and data were recorded after 90 seconds; all these procedures were followed-up by the clinician.

Serum component measurements
The blood stored in EDTA tube supernatants (5 cc of blood vein sample) was assayed to determine the levels of IL-8, tumor necrosis factor-α (TNF-α), and IL-6 via ELISA assay (Diaclone, France), following the manufacturer's instructions. At lower detection limits of 5, 4, and 2 pg/mL, the upper normal limitations were 62, 8.1, and 5.9 pg/mL, respectively. All the blood supernatants were stored at -70°С before assays. Total immunoglobulin (IgE; normal range < 165 IU/mL) was assayed by ELISA (EU-Immune, Germany). The complete blood cell count (CBC) with differentiation was also assayed. The level of superoxide dismutase (SOD) was assayed using the ELISA kit, with a normal range of 129-216 U/mL.

Glucocorticosteroid treatment responsiveness
The patients were categorized in 3 steps, based on the short-term step-up GINA strategy (12) until relieving the symptoms. The first step included inhaled corticosteroid

Clinical characteristics of uncontrolled asthma
A total of 246 uncontrolled asthma patients were enrolled in this study. The clinical characteristics and demographic profile of the subjects are presented in Table

Increased serum level of IL-8 in uncontrolled asthma patients compared to the control group
As illustrated in Figure 1, the serum IL-8 level in uncontrolled asthma patients (87.45 pg/mL; 5-7500) was significantly higher than that of the healthy controls ( However, no significant difference was found in these mediators between the groups.

Elevated serum IL-8 level indicative of the high incidence of uncontrolled asthma
In all uncontrolled asthma patients, the incidence of  Figure 2B).

Relationship between serum IL-8 level and blood neutrophils and airflow reversibility in uncontrolled asthma patients
There was a significant relationship between serum IL-  groups was significantly higher (P< 0.0001; Figure 3).  In  Figure 4B).   In general, IL-8 is a well-known type of classic chemokine, involved in neutrophil recruitment and activation (14). The increase in IL-8 level has been shown to play a pivotal role in different allergic or autoimmune disorders, such as allergic dermatitis (15), Henoch-Schonlein purpura (16), allergic rhinitis (17), allergic conjunctivitis (18), and autoimmune hepatitis (19,20). Search for a sensitive and convenient biomarker to identify uncontrolled asthmatics is an essential measure to improve asthma control. FeNO level was frequently measured to distinguish between airway eosinophilic and non-eosinophilic inflammation (24). The overall specificity was higher than its sensitivity, indicating a higher diagnostic potential for diagnosing rather than ruling out asthma (25). In this study, patients with a normal FeNO and elevated IL-8 were not uncommon. Generally, mere reliance on FeNO measurements may lead to the missed diagnosis of asthma and inadequate assessment, which is in line with the mentioned findings (25). Moreover, it is difficult and unrealistic to detect inflammatory mediators by inducing sputum or repeating bronchoscopy to evaluate asthma status.  Application of all auxiliary tests should be first based on the patient's complaints. In this study, wheezing, as a typical symptom of asthma, was only reported in half of the patients. During the study, we found different misdiagnoses in patients without wheezing symptoms. We also found that long-term heart palpitation might be a major complaint of asthma patients, which is worthy of attention in clinics.

A B
The limitations of the present study include the short observational time and incomplete data acquisition.
Moreover, no analysis of sputum was carried out to distinguish the cellular phenotype in patients. Therefore, it is not convincing to confirm the relationship between In conclusion, we found that the level and trend of increase in serum IL-8 level could be used as a preferable biomarker for uncontrolled asthma and glucocorticosteroid responsiveness in asthmatics (especially in a long period).
These findings can be helpful in distinguishing certain patient populations, who are more likely to respond to ICs.
This study also presents a theoretical framework for the development of new agents for IL-8 inhibition in order to provide more personalized and specialized asthma care for this subgroup of patients in the future.